Characterization and gene cloning of l-xylulose reductase involved in l-arabinose catabolism from the pentose-fermenting fungus Rhizomucor pusillus.

l-Xylulose reductase (LXR) catalyzes the reduction of l-xylulose to xylitol in the fungal l-arabinose catabolic pathway. LXR (RpLXR) was purified from the pentose-fermenting zygomycetous fungus Rhizomucor pusillus NBRC 4578. The native RpLXR is a homotetramer composed of 29 kDa subunits and preferred NADPH as a coenzyme.

Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.

Structural and computational analysis of peptide recognition mechanism of class-C type penicillin binding protein, alkaline D-peptidase from Bacillus cereus DF4-B.

Alkaline D-peptidase from Bacillus cereus DF4-B, called ADP, is a D-stereospecific endopeptidase reacting with oligopeptides containing D-phenylalanine (D-Phe) at N-terminal penultimate residue. ADP has attracted increasing attention because it is useful as a catalyst for synthesis of D-Phe oligopeptides or, with the help of substrate mimetics, L-amino acid peptides and proteins. Structure and functional analysis of ADP is expected to elucidate molecular mechanism of ADP.

Identification and characterization of D-xylulokinase from the D-xylose-fermenting fungus, Mucor circinelloides.

D-Xylulokinase catalyzes the phosphorylation of D-xylulose in the final step of the pentose catabolic pathway to form d-xylulose-5-phosphate. The D-xylulokinase activity was found to be induced by both D-xylose and L-arabinose, as well as some of the other enzymes involved in the pentose catabolism, in the D-xylose-fermenting zygomycetous fungus, Mucor circinelloides NBRC 4572. The putative gene, xyl3, which may encode D-xylulokinase, was detected in the genome sequence of this strain.

Molecular analysis of NAD⁺-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference.

The zygomycetous fungus Rhizomucor pusillus NBRC 4578 is able to ferment not only d-glucose but also d-xylose into ethanol. Xylitol dehydrogenase from R. pusillus NBRC 4578 (RpXDH), which catalyzes the second step of d-xylose metabolism, was purified, and its enzymatic properties were characterized.

Identification and characterization of D-xylose reductase involved in pentose catabolism of the zygomycetous fungus Rhizomucor pusillus.

Rhizomucor pusillus NBRC 4578 efficiently produces ethanol from lignocellulosic biomass because of its ability to ferment not only d-glucose, but also d-xylose. When the strain was cultivated on d-xylose, ethanol was gradually formed in the culture medium with a decrease in d-xylose and the simultaneous accumulation of xylitol, which suggested that the strain catabolized d-xylose with d-xylose reductase (XR) and xylitol dehydrogenase (XDH). XR (RpXR) was purified to homogeneity from the crude extract prepared from the mycelia of the strain grown on d-xylose.

Characterization and application of aminoamide-oxidizing enzyme from Aspergillus carbonarius AIU 205.

We isolated Aspergillus carbonarius AIU 205 as a new producer of an enzyme catalyzing oxidative deamination of 4-aminobutanamide (4-ABAD) to 4-oxobutanamide with the subsequent release of ammonia and hydrogen peroxide. Since the strain produced three enzymes with different Km values for 4-ABAD, the enzyme with lowest Km value (0.31 mM) was purified and revealed certain remarkable properties.

Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants.

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering.

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris.

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family.

High yield synthesis of 12-aminolauric acid by "enzymatic transcrystallization" of omega-laurolactam using omega-laurolactam hydrolase from Acidovorax sp. T31.

The genes encoding omega-laurolactam hydrolases from Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp.

The novel structure of a pyridoxal 5'-phosphate-dependent fold-type I racemase, alpha-amino-epsilon-caprolactam racemase from Achromobacter obae.

Alpha-amino-epsilon-caprolactam (ACL) racemase (ACLR) from Achromobacter obae catalyzes the interconversion of l- and d-ACL. ACLR belongs to the fold-type I group of pyridoxal 5'-phosphate (PLP) dependent enzymes. In this study, the first crystal structures of a fold-type I racemase are solved for the native form and epsilon-caprolactam-complexed form of ACLR at 2.

The screening, characterization, and use of omega-laurolactam hydrolase: a new enzymatic synthesis of 12-aminolauric acid.

Several omega-laurolactam degrading microorganisms were isolated from soil samples. These strains were capable of growing in a medium containing omega-laurolactam as sole source of carbon and nitrogen. Among them, five strains (T7, T31, U124, U224, and U238) were identified as Cupriavidus sp.

Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat).

A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.

Deduced catalytic mechanism of D-amino acid amidase from Ochrobactrum anthropi SV3.

D-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes D-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of D-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Omega loop, and a ground state with a disordered Omega loop.

Structures of D-amino-acid amidase complexed with L-phenylalanine and with L-phenylalanine amide: insight into the D-stereospecificity of D-amino-acid amidase from Ochrobactrum anthropi SV3.

The crystal structures of D-amino-acid amidase (DAA) from Ochrobactrum anthropi SV3 in complex with L-phenylalanine and with L-phenylalanine amide were determined at 2.3 and 2.2 A resolution, respectively.

New enzymatic method of chiral amino acid synthesis by dynamic kinetic resolution of amino acid amides: use of stereoselective amino acid amidases in the presence of alpha-amino-epsilon-caprolactam racemase.

D- and L-amino acids were produced from L- and D-amino acid amides by D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of alpha-amino-epsilon-caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides.

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins.

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.

L: -Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family.

Brevundimonas diminuta TPU 5720 produces an amidase acting L-stereoselectively on phenylalaninamide. The enzyme (LaaA(Bd)) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of approximately 53,000.

Purification, characterization, gene cloning and nucleotide sequencing of D: -stereospecific amino acid amidase from soil bacterium: Delftia acidovorans.

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively.

A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for beta-alanyl dipeptides.

We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene, ramA, from the Pseudomonas sp. MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 (43% identity). The DmpA (called L-aminopeptidase D-Ala-esterase/amidase) hydrolyzes alanine-p-nitroanilide, alaninamide, and alanine methylester with a preference for the D-configuration of the alanine, whereas the enzyme acts as an L-stereoselective aminopeptidase on a tripeptide Ala-(Gly)2, indicating a reverse stereoselectivity [Fanuel L, Goffin C, Cheggour A, Devreese B, Van Driessche G, Joris B, Van Beeumen J & Frère J-M (1999) Biochem J341, 147-155].

A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide.

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase.

S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase.

An amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas azotoformans IAM 1603 and characterized. The enzyme acted S-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (S)-piperazine-2-carboxylic acid. N-terminal and internal amino acid sequences of the enzyme were determined.

Genes for an alkaline D-stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genome.

Alkaline D-peptidase (Adp) from Bacillus cereus DF4-B is a D-stereospecific endopeptidase acting on oligopeptides composed of D-phenylalanine and the primary structure deduced from its gene, adp, shows a similarity with D-stereospecific hydrolases from Ochrobactrum anthropi strains. We have isolated DNA fragments covering the flanking region of adp from DF4-B genome and found an additional gene, adp2, located upstream of adp. The deduced amino acid sequence of Adp2 showed 96% and 85% identity with those of Adp from B.